1. Description of the Invention
The present invention is concerned with the identification, detection and isolation of a novel autonomously replicating biological particle. The biological particle is a bacteria-like organism that readily passes through sterile filters of pore size of 0.3 .mu.m. It is a member of a novel genus hereby referred to as Nanobacterium and has been accorded a special name.
2. Description of the Prior Art
It is generally believed that animal blood and tissues, except those facing external surfaces, such as the skin and mucosa, are sterile. This idea is primarily based on microbiological culture experiments utilizing culture media optimized for the culture of pathogenic bacteria generally associated with humans and animals. By utilizing special culture media, however, cell-wall defective bacterial variants (oftentimes referred to as bacterial L-forms) have been identified from a variety of living material. Bacterial L-forms have been detected using special culture media in approximately fifty percent of both healthy and sick humans. Conventional culture methods do not support the growth of those microbes and the possible biological significance of their presence in blood and tissues is not known at this time.
The special bacterial L-form culture medium is typically a hyperosmolar medium containing a sugar such as sucrose, sodium chloride or polyvinylpyrrolidone (PVP) as a stabilizer; brain heart infusion broth or other bacteriological nutrient source; agar; protein such as horse serum; and an antibiotic such as penicillin. Penicillin and other antibiotics prevent peptidoglycan synthesis which both convert normal bacteria to L-forms and prevent the reversion of L-forms back to cell wall containing bacteria.
Animal or human serum is widely utilized in cell culture as a growth supporter. Serum for cell culture is collected from animals at slaughterhouses. Animal serum has been recognized as a major source of contamination in cell culture. For instance, mycoplasma contamination was very common before commercial cell culture serum was screened for the presence of mycoplasma.
Animal or human serum is sterilized using a step-wise sterile filtration procedure at high porosity. The final and smallest filter size is generally in the range of about 0.22 .mu.m to 0.1 .mu.m. Common bacteria are retained by 0.45 .mu.m filters. The sterility of the final product is typically detected by taking samples from the serum lot and incubating them at 25.degree. C. and 37.degree. C. for one or two days and then performing standard microbiological culture assays on agar media or filters. The cultures are then examined at intervals up to two weeks. The conventional culture method will detect bacteria growing under culture conditions but will not show fastidious or noncolony forming bacteria.
Bacterial L-forms or cell wall defective bacteria have been recognized for some time. Such bacteria have been found in many types of organisms including humans. Bacterial L-forms readily pass through sterile filtration. Thus, since bacterial L-forms may be present in animal sera and animal sera are often used in cell cultures and blood products, the bacterial L-forms remain in those compositions since they are usually sterilized by filtration.
The presence of bacterial L-forms in cell cultures and inside cultured cells, and cell morphology during such an infection have been studied by several authors. These studies were carried out by inoculating cultured cells with artificially produced bacterial L-forms. Only one published report describes a cell culture being contaminated by something resembling a bacterial L-form originating from nature, that is, outside the laboratory. That report by I. Willers, S. Singh, K. R. Held, and H. W. Goedde, "High HPRT Activity in Fibroblasts from Patients with Lesch-Nyhan Syndrome due to Bacterial `L-form` Contamination", Adv. Exp. Med. Biol., Vol. 122, pp. 327-331, 1980) discloses that fibroblasts from a Less-Nyhan patient were positive for HPRT enzyme activity. This enzyme activity is totally absent from Less-Nyhan fibroblasts, and thus the contaminants caused an erroneous result in testing of this biochemical marker of importance for genetic counseling. This report is also the only publication indicating that cell culture serum is a source for L-form infection. However, the organisms were neither identified nor was their nature provided in any detail.
Bacteriological sterility of cell cultures is an absolute requirement for metabolic as well as other types of experiments in order to obtain valid results. Bacteria smaller than the porosity of filters used for sterilization or bacteria having elastic cell walls (L-forms) can readily pass through the filter and thus prevent adequate sterilization. If the contaminating organisms are not detected by the methods used for confirming bacteriological sterility, the product, e.g., the sera, is likely to form a very potent hazard for cell culture experiments. The available bacteriological methods are insufficient for the detection of such contamination.
The present inventor has successfully isolated and cultivated a new bacterial contaminant in substantially pure form by developing new methods for cultivating and detecting such filterable bacteria discovered from commercial cell culture sera.